Abstract The 2014-15 Ebola virus (EBOV) outbreak in West Africa highlighted the urgent need for the development of antivirals to treat or prevent filovirus infections. The outbreak was associated with nearly 30,000 suspected or confirmed cases and more than 11,000 documented deaths. While pathogenesis of Ebola virus disease (EVD) is complex and incompletely understood, high levels of virus are found in the blood throughout the course of the disease, indicating that viral load plays an important role in pathogenesis. This suggests that antivirals targeting viral proteins could have a significant therapeutic effect, even at late stages. While some attempts have been made at screening for small molecule EBOV inhibitors, these efforts have been hampered by the lack of detailed biochemical and molecular knowledge required for design of target-specific HTS assays. Another obstacle is the low throughput of BSL-4 viral assays that otherwise could be used to screen large libraries for compounds with antiviral activity. Particularly lacking are direct-acting antivirals that inhibit viral protein function, making this an understudied area of investigation considering the severity of the disease and the potential for devastating outbreaks. In this application it is proposed to address four important potential viral targets, and test the hypothesis that they can be targeted with small molecule inhibitors. In Aim 1, inhibitors of VP24, VP35, VP40 and NP will be identified using a yeast-based platform that identifies chemical suppressors of viral protein activity. These compounds will also be tested for antiviral activity using two independent assays. In Aim 2, antiviral compounds will be tested for direct binding to their target using TSA and ITC, in order to identify the most attractive, direct-acting inhibitors for future medicinal chemistry and structure based development.